Protein-lysine acetylation is a major post-translational modification, where acetyl-proteomic studies have catalogued thousands of acetylation sites, representing proteins in diverse cellular pathways. Mechanistic studies have revealed diverse consequences of specific protein acetylation, but such functional studies have lagged behind protein cataloguing. Deacetylases and acetyl-CoA (AcCoA) dependent transferases are implicated in controlling the acetylation state of target proteins, though the dynamics and the mechanisms that lead to cellular compartment-specific acetylation is unclear. While evidence of enzyme-catalyzed acetylation exits in the cytoplasm, nucleus and ER, most mitochondrial protein acetylation is thought to be uncatalyzed. We recently described rapid cellular protein acetylation in response to growth factor stimulation, which remarkably includes a subset of mitochondrial proteins, indicative of enzyme-catalyzed acetylation. Thus, the contribution of non-enzymatic acetylation, driven by AcCoA flux/levels, and of enzyme-catalyzed acetylation remains poorly understood. With strategic use of cultured cell lines and mouse models, a major portion of this proposal investigates the mechanisms that control specific and proteome-wide acetylation in both acute cellular responses and chronic energy-depleted conditions. In addition to cellular mechanisms that promote acetylation, regulation of deacetylases drive functional consequences in various organelles. The NAD+-dependent protein deacetylases (SIRT1-7) are a major family of enzymes found in diverse sub-cellular compartments. Mitochondrial SIRT3 and nuclear SIRT6 and SIRT7 are the subject of this proposal. SIRT3 deacetylates and increases the catalytic efficiency of enzymes involved in oxidative metabolism. SIRT6 and SIRT7 are chromatin bound proteins that can remove specific lysine acetylations on histones. The mechanisms by which SIRT6 and SIRT7 catalyze the deacetylation of nucleosomes are completely unknown, yet the genetic and biological importance of these proteins is advancing rapidly. Loss-of-function (deacetylation) mutants of SIRT6 cause cancer and perinatal lethality. Our recent data on SIRT6 provides unprecedented insight into the molecular functions of SIRT6 and reveal the therapeutic potential of small-molecule activation. Our preliminary data on SIRT7 indicates novel activities related to nucleosome binding. To address these major gaps in our understanding of sirtuin biology and of the mechanisms that drive functionally relevant protein acetylation in a compartment-specific manner, the following aims are proposed: Aim 1, Determine the mechanisms of nucleosomal deacetylation by SIRT6 and SIRT7; Aim 2, Elucidate the mechanisms of compartment-specific protein acetylation under acute stimulation and chronic metabolic stress; and Aim 3, Reveal pathway-level and site-specific functional roles of protein acetylation in energy metabolism and mitochondrial proteostasis.